Variant aim

ABSTRACT

The present invention relates to the provision of a recombinant AIM that maintains functions, does not multimerize, and is not inactivated by IgM (e.g., a protein containing an amino acid sequence wherein cysteine at amino acid number 168 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid, and the like).

TECHNICAL FIELD

The present invention relates to a mutant AIM, a nucleic acid encodingthe mutant AIM, an expression vector containing the nucleic acid, a hostcell containing the expression vector, a production method of a mutantAIM using the host cell, and a pharmaceutical composition containing themutant AIM.

BACKGROUND ART

AIM (apoptosis inhibitor of macrophage, or CD5L) is a factor which isspecifically produced by a macrophage identified by the presentinventors and suppresses apoptosis of the macrophage itself (non-patentdocument 1), and association with several diseases has been suggested sofar. For example, the blood concentration of AIM increases with obesity,and AIM is taken up by adipocytes due to CD36-mediated endocytosis andinduces lipolysis of accumulated neutral fats, which suggestsrelationship with antiobesity (non-patent document 2). AIM releases freefatty acid from adipocytes by lipolysis of neutral fats, and thereleased fatty acid induces/maintains chronic inflammation in adiposetissue via stimulation of toll-like receptors. Metabolic syndrome isbased on the acquisition of insulin resistance associated with obesity.Since chronic inflammation in adipose tissue is important, AIM is saidto be associated with metabolic syndrome (non-patent document 3). Thepresent inventors also clarified that AIM suppresses the differentiationof fat progenitor cells into mature adipocytes and induces thedecomposition of fat droplets in adipocytes, and reported thepossibility of application of AIM to obesity (patent document 1).Furthermore, the present inventors clarified that obese AIM knockout(KO) mice loaded with a high-calorie diet show pathology similar tohuman NASH pathology, such as obesity, fatty liver, fibrosis of liverparenchyma, and carcinogenesis, and reported the possibility ofapplication of AIM to liver diseases (patent document 2). In addition,the present inventors clarified that AIM KO mice that underwentbilateral transient renal ischemia reperfusion developed acute renalfailure, followed by the accumulation of necrotic renal tubular cellsand the accompanying rapid progression of renopathy, and exacerbation ofthe systemic condition, and a high frequency of death was confirmed. Theinventors showed that when AIM was administered to the AIM KO mice, BUNlevel was improved, renal function was rapidly improved, and systemicsymptoms and mortality were also improved, and reported the possibilityof the treatment of acute renal failure and the prophylaxis or treatmentof chronic renal diseases by the supplementation of AIM (patent document3). Also, the inventors reported that in blood, AIM forms a complex withIgM pentamer in the Fc region, which protects AIM from renal filtrationand maintains high level of blood AIM (non-patent documents 4, 5).

Generally, when a protein having a pharmacological effect is used as apharmaceutical product, it is necessary to prepare a uniform proteinthat satisfies the conditions such as maintenance of function, absenceof coagulation and the like. A typical method for preparing a protein isa method including culturing a cell into which an expression vectorhaving a nucleic acid encoding a desired protein has been introduced,and isolating and purifying a recombinant protein secreted inside thecell or secreted into the culture medium from the cell. In the presentmethod, Escherichia coli is preferably used as a host cell because it iseasy to handle in gene transfer, culturing and the like. However,problems may occur during preparation of protein under conditionsdifferent from the original environment. For example, eukaryote-derivedproteins may form intramolecular disulfide bonds between cysteines whenforming higher-order structures. When such a protein is prepared bytransforming Escherichia coli, the formation of disulfide bond isprevented since the cytoplasm of Escherichia coli is under strongreducing conditions. As a result, problems may occur in that theoriginal higher-order structure is not reproduced, the purifiedrecombinant protein loses its original function or exhibits toxicity,and the like. When a mammalian cell is used as a host cell and thetarget protein is expressed at a concentration higher than that at whichthe protein is no/many present in the cell, the recombinant proteins mayform a multimer by disulfide bond, which may affect purification of therecombinant protein or pharmacokinetics in vivo. Since AIM contains 3scavenger receptor cysteine-rich (SRCR) domains consisting of about 100amino acids containing many cysteines, Escherichia coli is not suitableas a host cell when recombinant AIM is produced, and even when mammaliancells are used, it was necessary to verify whether or not therecombinant AIM is multimerized, and when multimerization is confirmed,a means for preventing the multimerization needs to be considered.However, no report has been made to date as regards the multimerizationof recombinant AIM. Furthermore, since AIM that formed a complex withIgM pentamer becomes inactive, when recombinant AIM is administered as apharmaceutical product, it is necessary to control the formation of acomplex of AIM and IgM pentamer. This problem remains unsolved.

DOCUMENT LIST Patent Documents

-   patent document 1: WO 2010/140531-   patent document 2: WO 2013/162021-   patent document 3: WO 2015/119253

Non-Patent Documents

-   non-patent document 1: T. Miyazaki et al., J Exp Med. 189:413-422,    1999-   non-patent document 2: J. Kurokawa et al., Cell Metab. 11:479-492,    2010-   non-patent document 3: J. Kurokawa, Proc Natl Acad Sci USA.,    108:12072-12077, 2011-   non-patent document 4: S. Arai et al., Cell Rep. 3:1187-1198, 2013-   non-patent document 5: T. Miyazaki et al., Cell Mol Immunol.    15:562-574, 2018

SUMMARY OF INVENTION Technical Problem

The present invention aims to provide a recombinant AIM that maintainsfunctions, does not multimerize, and is not inactivated by IgM.

Solution to Problem

The present inventors confirmed that a multimer is formed when awild-type human recombinant AIM is expressed using HEK293, and focusedon cysteine contained in human AIM as a cause of multimerization. Thepresent inventors predicted from the three-dimensional structureprediction of human AIM that the cysteine at amino acid number 168 andthe cysteine at amino acid number 277 of wild-type human AIM shown inSEQ ID NO: 1 are involved in the multimer formation. It was confirmedthat the mutant human recombinant AIM does not multimerize when a mutanthuman recombinant AIM in which the cysteine is substituted with serineis prepared. The recovery efficiency of the mutant recombinant AIMincreased as compared to that of the wild-type recombinant AIM.Furthermore, the mutant human recombinant AIM maintained the endocytoticactivity on macrophages, which is one of the functions of wild-typehuman AIM. Surprisingly, it was confirmed that the activity wassignificantly improved in AIM in which cysteine of amino acid number 168of wild-type human AIM shown in SEQ ID NO: 1 was replaced with serine.It was also confirmed that the wild-type mouse recombinant AIM forms adimer, and it was also found that the cysteine at amino acid number 168of the wild-type mouse AIM shown in SEQ ID NO: 2 is the cause of dimerformation. Furthermore, the mutant human AIM and the mutant mouse AIM inwhich the cysteine at amino acid number 168 of wild-type human AIM andthe cysteine at amino acid number 168 of wild-type mouse AIM arerespectively substituted with serine do not form a complex with the IgMpentamer. The present inventors have conducted further studies based onthese findings and completed the present invention.

That is, the present invention provides

[1] a mutant human AIM comprising an amino acid sequence of any one ofthe following (1) to (5) and having an activity of wild-type human AIM(1) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid,(2) an amino acid sequence wherein cysteine at amino acid number 277 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid,(3) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid, and an amino acid sequence wherein cysteine at aminoacid number 277 of the amino acid sequence shown in SEQ ID NO: 1 issubstituted with another amino acid,(4) an amino acid sequence substantially the same as the amino acidsequence of any one of (1) to (3), and cysteines present in the aminoacid sequence of any one of (1) to (3) and the substituted another aminoacid remain,(5) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of any one of (1) to (3) and the substituted another aminoacid;[2] the mutant human AIM of [1], wherein the another amino acid is anamino acid selected from the group consisting of asparagine, glutamine,serine, and threonine;[3] the mutant human AIM of [1], wherein the another amino acid isserine;[4] a mutant mouse AIM comprising an amino acid sequence of any one ofthe following (1) to (3) and having an activity of wild-type mouse AIM(1) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 2 is substituted withanother amino acid,(2) an amino acid sequence substantially the same as the amino acidsequence of (1), and cysteines present in the amino acid sequence of (1)and the substituted another amino acid remain,(3) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of (1) and the substituted another amino acid;[5] the mutant mouse AIM of [4], wherein the another amino acid is anamino acid selected from the group consisting of asparagine, glutamine,serine, and threonine;[6] the mutant mouse AIM of [4], wherein the another amino acid isserine;[7] a nucleic acid comprising the base sequence of the following (1) or(2)(1) a base sequence encoding the mutant human AIM of any one of [1] to[3],(2) a base sequence encoding the mutant mouse AIM of any one of [4] to[6];[8] an expression vector comprising the nucleic acid of [7];[9] a host cell comprising the expression vector of [8];[10] a method for producing a mutant human AIM or a mutant mouse AIM,comprising culturing the host cell of [9];[11] a pharmaceutical composition comprising a mutant AIM of thefollowing (1) or (2)(1) the mutant human AIM of any one of [1] to [3],(2) the mutant mouse AIM of any one of [4] to [6];and the like.

Advantageous Effects of Invention

The mutant AIM of the present invention has the function equivalent tothat of the wild-type recombinant AIM, or has an improved function, andis characterized in that it does not multimerize when expressed as arecombinant AIM. By the absence of multimerization, recombinant AIM doesnot precipitate by insolubilization and, as a result, the recovery rateof recombinant AIM is improved and the risk associated with in vivoadministration is avoided. Furthermore, the mutant AIM of the presentinvention has a feature that, when administered to a living body, itdoes not form a complex with an IgM pentamer and, as a result, is notinactivated. Therefore, a decrease in the titer of the administeredmutant AIM can be prevented and the dose can be reduced.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an electrophoretic image of wild-type human rAIM afterpurification and concentration. Monomer: monomer, Dimer: dimer, Polymer:multimer. The dark-colored part at the top of the gel (indicated by anarrow) is a multimer having a huge molecular weight.

FIG. 2 shows an electrophoretic image of wild-type mouse rAIM afterpurification and concentration. Monomer: monomer, Dimer: dimer.

FIG. 3 shows the amino acid sequences of wild-type, 2CS, 3CS and 2/3CShuman AIMs. The underlined part indicates the hinge connecting the SRCRdomains.

FIG. 4 shows a three-dimensional structure of wild-type human AIM.

FIG. 5 shows a three-dimensional structure of wild-type mouse AIM.

FIG. 6 shows the amino acid sequences of wild-type and 2CS mouse AIMS.The underlined part indicates the hinge connecting the SRCR domains.

FIG. 7 shows electrophoretic images of wild-type, 2CS, 3CS and 2/3CShuman rAIMs after purification and concentration. Monomer: monomer,Dimer: dimer, Polymer: multimer. The other bands are contamination withnon-specific proteins other than AIM.

FIG. 8 shows an electrophoretic image of wild-type and 2CS mouse rAIMsafter purification and concentration. Monomer: monomer, Dimer: dimer.

FIG. 9 is a drawing showing uptake of rAIM by peritoneal macrophagecells. The vertical axis shows fluorescence intensity.

FIG. 10 shows a Western blot image (A) of proteins obtained from culturesupernatant containing wild-type or 2CS mouse rAIM and mouse IgMpentamer, and a Western blot image (B) of proteins obtained from culturesupernatant containing wild-type or 2CS human rAIM and human IgMpentamer. a-mAIM: anti-mouse AIM, a-hAIM: anti-human AIM, a-mIgM:anti-mouse IgM, a-hIgM: anti-human IgM.

DESCRIPTION OF EMBODIMENTS

The present invention provides a mutant human AIM comprising an aminoacid sequence of any one of the following (1a) to (5a) and having anactivity of wild-type human AIM (hereinafter to be also referred to asthe mutant human AIM of the present invention)

(1a) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid,(2a) an amino acid sequence wherein cysteine at amino acid number 277 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid,(3a) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid, and an amino acid sequence wherein cysteine at aminoacid number 277 of the amino acid sequence shown in SEQ ID NO: 1 issubstituted with another amino acid,(4a) an amino acid sequence substantially the same as the amino acidsequence of any one of (1a) to (3a), and cysteines present in the aminoacid sequence of any one of (1a) to (3a) and the substituted anotheramino acid remain,(5a) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of any one of (1a) to (3a) and the substituted another aminoacid.

In addition, the mutant human AIM of the present invention preferablycontains the amino acid sequence of any one of the following (1b) to(5b).

(1b) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted with serine(SEQ ID NO: 3),(2b) an amino acid sequence wherein cysteine at amino acid number 277 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted with serine(SEQ ID NO: 4),(3b) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withserine, and an amino acid sequence wherein cysteine at amino acid number277 of the amino acid sequence shown in SEQ ID NO: 1 is substituted withserine (SEQ ID NO: 5),(4b) an amino acid sequence substantially the same as the amino acidsequence of any one of (1b) to (3b), and cysteines present in the aminoacid sequence of any one of (1b) to (3b) and the substituted serineremain,(5b) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of any one of (1b) to (3b) and the substituted serine.

In the present invention, the amino acid sequence shown in SEQ ID NO: 1is the amino acid sequence of wild-type human AIM. The wild-type humanAIM is a protein mainly present in blood containing three cysteine-richScavenger-Receptor Cysteine-Rich (SRCR) domains. The SRCR1 domain, SRCR2domain, and SRCR3 domain contained in wild-type human AIM contain 8, 9,and 9 cysteines, respectively. Of these cysteines, 8, 8, and 8 cysteinesrespectively contribute to formation intramolecular disulfide bond andwild-type human AIM forms a higher-order structure. The cysteines thatare not involved in the formation of higher-order structure of wild-typehuman AIM in vivo are the cysteine at amino acid number 168 and thecysteine at amino acid number 277 in SEQ ID NO: 1. These two cysteinespresent in wild-type human AIM do not contribute to the intramoleculardisulfide bond, but instead form an intermolecular disulfide bondbetween recombinant human AIMS. As a result, recombinant human AIMmultimerizes and precipitates as an insolubilized protein. Furthermore,the above-mentioned cysteine at amino acid number 168 of SEQ ID NO: 1forms a complex consisting of wild-type human AIM and human IgM pentamerby forming an intermolecular disulfide bond with a particular cysteinein the IgM pentamer and inactivates wild-type human AIM.

As mentioned above, the mutant human AIM of the present inventioncontains the amino acid sequence of any of the following (1a) to (3a).

(1a) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid.(2a) an amino acid sequence wherein cysteine at amino acid number 277 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid.(3a) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid, and an amino acid sequence wherein cysteine at aminoacid number 277 of the amino acid sequence shown in SEQ ID NO: 1 issubstituted with another amino acid.

The recombinant mutant human AIM of the present invention preventsformation of intermolecular disulfide bond between recombinant humanAIMs by substituting cysteine at amino acid number 168 and/or cysteineat amino acid number 277 in the amino acid sequence shown in SEQ ID NO:1 with different amino acids, whereby multimerization of recombinanthuman AIM can be prevented. In addition, it prevents formation ofintermolecular disulfide bond between recombinant human AIM and IgMpentamer by substituting cysteine at amino acid number 168 in the aminoacid sequence shown in SEQ ID NO: 1 with a different amino acid, wherebyinactivation of recombinant human AIM can be prevented. The differentamino acids used for substitution of the above-mentioned cysteine atamino acid number 168 and/or cysteine at amino acid number 277 in theamino acid sequence shown in SEQ ID NO: 1 is not particularly limited aslong as the obtained mutant human AIM can maintain or improve theactivities similar to those of the wild-type human AIM (here, “activity”means, for example, endocytosis activity to macrophages, apoptosissuppressive activity of macrophages, arteriosclerosismaintenance/promotion activity, adipocyte differentiation suppressiveactivity, adipocyte lipolytic activity, adipocyte reducing activity,CD36 binding activity, adipocyte endocytosis activity, FAS bindingactivity, FAS function suppressive activity, antiobesity activity, liverdiseases (fatty liver, NASH, liver cirrhosis, liver cancer), preventiveor therapeutic activity of kidney diseases (acute renal failure, chronicnephritis, chronic renal failure, nephrotic syndrome, diabeticnephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy,nephropathy associated with collagen disease or IgM nephropathy), and anamino acid showing similar physicochemical properties as cysteine ispreferably mentioned. The different amino acids for substituting theabove-mentioned cysteine at amino acid number 168 and the differentamino acids for substituting cysteine at amino acid number 277 in theamino acid sequence shown in SEQ ID NO: 1 may be the same or different.Examples of the amino acid similar to cysteine include amino acidsclassified as polar neutral amino acids, and specifically include aminoacids selected from the group consisting of asparagine, glutamine,serine, and threonine. Among these, serine is preferred from theviewpoint of structural similarity. Therefore, the mutant human AIM ofthe present invention containing any of the amino acid sequences of theabove-mentioned (1a) to (3a) may preferably be a mutant human AIMcontaining any of the following amino acid sequences.

(1b) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted with serine(SEQ ID NO: 3).(2b) an amino acid sequence wherein cysteine at amino acid number 277 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted with serine(SEQ ID NO: 4).(3b) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withserine, and an amino acid sequence wherein cysteine at amino acid number277 of the amino acid sequence shown in SEQ ID NO: 1 is substituted withserine (SEQ ID NO: 5).

Particularly, the endocytic activity of (1b) a mutant human AIMcontaining the amino acid sequence (SEQ ID NO: 3) in which the cysteineat amino acid number 168 of the amino acid sequence shown in SEQ ID NO:1 is substituted with serine is higher than the activity of wild-typehuman AIM as shown in the below-mentioned Examples and the function asAIM is suggested to be more superior.

The mutant human AIM of the present invention may contain the amino acidsequence of the following (4a) or (5a) in place of the amino acidsequence of any of the above-mentioned (1a) to (3a).

(4a) an amino acid sequence substantially the same as the amino acidsequence of any one of (1a) to (3a), and cysteines present in the aminoacid sequence of any one of (1a) to (3a) and the substituted anotheramino acid remain,(5a) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of any one of (1a) to (3a) and the substituted another aminoacid.

The mutant human AIM of the present invention may contain the amino acidsequence of the following (4b) or (5b) in place of the amino acidsequence of any of the above-mentioned (1b) to (3b).

(4b) an amino acid sequence substantially the same as the amino acidsequence of any one of (1b) to (3b), and cysteines present in the aminoacid sequence of any one of (1b) to (3b) and the substituted serineremain.(5b) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of any one of (1b) to (3b) and the substituted serine.

Specific examples of the amino acid sequence substantially the same asthe above-mentioned amino acid sequence of any one of (1a) to (3a) (orany one of the above-mentioned (1b) to (3b)), in which cysteines presentin the amino acid sequence of any one of the above-mentioned (1a) to(3a) (or the above-mentioned (1b) to (3b)) and the substituted anotheramino acid (preferably, the substituted another amino acid is serine)remain include an amino acid in which cysteines at amino acid numbers10, 26, 39, 44, 73, 83, 91, 101, 124, 140, 153, 158, 168, 185, 195, 205,215, 230, 246, 259, 264, 277, 292, 302, 312 and 322 of the amino acidsequence of any one of the above-mentioned (1a) to (3a) (or any one ofthe above-mentioned (1b) to (3b)), and the substituted another aminoacids (preferably, the substituted another amino acid is serine) are notchanged, and the amino acid sequence parts other than the amino acidshave a homology of not less than about 85%, preferably not less thanabout 90%, further preferably not less than about 95%, most preferablynot less than about 98%, with the amino acid sequence shown in SEQ IDNO: 1 and the like. As used herein, the “homology” means the proportion(%) of the same amino acid residues and similar amino acid residuesrelative to the total overlapping amino acid residues, in an optimalalignment (preferably, the algorithm is capable of consideringintroduction of gap into one of or both of the sequences for optimalalignment), when two amino acid sequences are aligned using amathematical algorithm known in the technical field. The “similar aminoacid” means amino acids having similar physicochemical properties and,for example, amino acids classified in the same group such as aromaticamino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val),polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidicamino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr),amino acids having a small side chain (Gly, Ala, Ser, Thr, Met) and thelike can be mentioned. It is predicted that the substitution with suchsimilar amino acids does not change the phenotype of the protein (thatis, conservative amino acid substitution). Specific examples of theconservative amino acid substitution are well known in the technicalfield and are described in various documents (e.g., Bowie et al.,Science, 247: 1306-1310 (1990)).

The homology of the amino acid sequences in the present specificationcan be calculated using homology calculation algorithm NCBI BLAST(National Center for Biotechnology Information Basic Local AlignmentSearch Tool) and under the following conditions (expectancy=10; gapallowed; matrix=BLOSUM62; filtering=OFF). Examples of other algorithmfor determining the homology of the amino acid sequence include thealgorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [said algorithm is incorporated in the NBLAST andXBLAST program (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol.Biol., 48: 444-453 (1970) [said algorithm is incorporated in the GAPprogram in the GCG software package], the algorithm described in Myersand Miller, CABIOS, 4: 11-17 (1988) [said algorithm is incorporated inALIGN program (version2.0) which is a part of the CGC sequence alignmentsoftware package], the algorithm described in Pearson et al., Proc.Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [said algorithm isincorporated in the FASTA program in the GCG software package] and thelike, and these can also be similarly used preferably.

More preferably, examples of the amino acid sequence substantially thesame as the above-mentioned amino acid sequence of any one of (1a) to(3a) (or any one of the above-mentioned (1b) to (3b)), in whichcysteines present in the amino acid sequence of any one of theabove-mentioned (1a) to (3a) (or the above-mentioned (1b) to (3b)) andthe substituted another amino acid (preferably, the substituted anotheramino acid is serine) remain include an amino acid which cysteines atamino acid numbers 10, 26, 39, 44, 73, 83, 91, 101, 124, 140, 153, 158,168, 185, 195, 205, 215, 230, 246, 259, 264, 277, 292, 302, 312 and 322of the amino acid sequence of any one of the above-mentioned (1a) to(3a) (or any one of the above-mentioned (1b) to (3b)), and thesubstituted another amino acids (preferably, the substituted anotheramino acid is serine) are not changed, and the amino acid sequence partsother than the amino acids have an identity of not less than about 85%,preferably not less than about 90%, further preferably not less thanabout 95%, most preferably not less than about 98%, with the amino acidsequence shown in SEQ ID NO: 1.

The position other than cysteines present in the amino acid sequence ofany one of the above-mentioned (1a) to (3a) (or the above-mentioned (1b)to (3b)) and the substituted another amino acid (preferably, thesubstituted another amino acid is serine) is the position other than theamino acid numbers 10, 26, 39, 44, 73, 83, 91, 101, 124, 140, 153, 158,168, 185, 195, 205, 215, 230, 246, 259, 264, 277, 292, 302, 312 and 322of the amino acid sequence of any one of the above-mentioned (1a) to(3a) (or any one of the above-mentioned (1b) to (3b)), and is notparticularly limited as long as it is a position at which a mutant humanAIM containing deletion, addition, insertion or substitution, or acombination thereof can maintain or improve the activities the wild-typehuman AIM has. The number of deletion, addition, insertion orsubstitution, or a combination thereof is one to several, preferably, 1to 5, 1 to 4, 1 to 3, or 1 or 2. The amino acids to be used forsubstitution may be similar to the above-mentioned amino acids.

The present invention also provides a mutant mouse AIM containing anamino acid sequence of any one of the following (1c) to (3c) and havingan activity of wild-type mouse AIM (hereinafter to be also referred toas the mutant mouse AIM of the present invention)

(1c) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 2 is substituted withanother amino acid,(2c) an amino acid sequence substantially the same as the amino acidsequence of (1c), and cysteines present in the amino acid sequence of(1c) and the substituted another amino acid remain,(3c) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of (1c) and the substituted amino acid.

In addition, the mutant mouse AIM of the present invention preferablycontains the amino acid sequence of any one of the following (1d) to(3d).

(1d) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 2 is substituted with serine(SEQ ID NO: 6),(2d) an amino acid sequence substantially the same as the amino acidsequence of (1d), and cysteines present in the amino acid sequence of(1d) and the substituted serine remain,(3d) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of (1d) and the substituted serine.

In the present invention, the amino acid sequence shown in SEQ ID NO: 2shows the amino acid sequence of wild-type mouse AIM. Similar to thewild-type human AIM, the wild-type mouse AIM contains 3 SRCR domains.Cysteines contained in the SRCR1 domain, SRCR2 domain and SRCR3 domainare, unlike wild-type human AIM, 8, 9 and 8, respectively. The cysteinethat is not involved in the formation of higher-order structure ofwild-type mouse AIM in vivo is only the cysteine at amino acid number168 in SEQ ID NO: 2. This cysteine present in wild-type mouse AIM doesnot contribute to the intramolecular disulfide bond, but instead formsan intermolecular disulfide bond between recombinant mouse AIMs. As aresult, recombinant mouse AIM dimerizes. Furthermore, theabove-mentioned cysteine at amino acid number 168 of SEQ ID NO: 2 formsa complex consisting of wild-type mouse AIM and mouse IgM pentamer byforming an intermolecular disulfide bond with a particular cysteine inthe IgM pentamer and inactivates wild-type mouse AIM.

As mentioned above, the mutant mouse AIM of the present inventioncontains (1c) amino acid sequence wherein cysteine at amino acid number168 of the amino acid sequence shown in SEQ ID NO: 2 is substituted withanother amino acid.

The mutant mouse AIM of the present invention prevents formation ofintermolecular disulfide bond between recombinant mouse AIMs bysubstituting cysteine at amino acid number 168 in the amino acidsequence shown in SEQ ID NO: 2 with a different amino acid, wherebydimerization of recombinant mouse AIM can be prevented. In addition, itprevents formation of intermolecular disulfide bond between recombinantmouse AIM and IgM pentamer by substituting cysteine at amino acid number168 in the amino acid sequence shown in SEQ ID NO: 2 with a differentamino acid, whereby inactivation of recombinant mouse AIM can beprevented. The different amino acids used for substitution of theabove-mentioned cysteine at amino acid number 168 in the amino acidsequence shown in SEQ ID NO: 2 is not particularly limited as long asthe obtained mutant mouse AIM can maintain or improve the activitiessimilar to those of the wild-type mouse AIM (here, “activity” means thesame as in wild-type human AIM). An amino acid showing physicochemicalproperties similar to those of cysteine is preferred. Examples of theamino acid similar to cysteine include amino acids classified as polarneutral amino acids, and specifically include amino acids selected fromthe group consisting of asparagine, glutamine, serine, and threonine.Among these, serine is preferred from the viewpoint of structuralsimilarity. Therefore, the above-mentioned mutant mouse AIM of thepresent invention is preferably mutant mouse AIM containing (1d) anamino acid sequence wherein cysteine at amino acid number 168 of theamino acid sequence shown in SEQ ID NO: 2 is substituted with serine(SEQ ID NO: 6).

The mutant mouse AIM of the present invention may contain the amino acidsequence of the following (2c) or (3c) in place of the amino acidsequence of the above-mentioned (1c).

(2c) an amino acid sequence substantially the same as the amino acidsequence of (1c), and cysteines present in the amino acid sequence of(1c) and the substituted another amino acid remain,(3c) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of (1c) and the substituted amino acid.

The mutant mouse AIM of the present invention may also contain the aminoacid sequence of the following (2d) or (3d) in place of the amino acidsequence of the above-mentioned (1d).

(2d) an amino acid sequence substantially the same as the amino acidsequence of (1d), and cysteines present in the amino acid sequence of(1d) and the substituted serine remain,(3d) an amino acid sequence further comprising deletion, addition,insertion or substitution of one to several amino acids or a combinationthereof at a position other than cysteines present in the amino acidsequence of (1d) and the substituted serine.

Specific examples of the amino acid sequence substantially the same asthe above-mentioned amino acid sequence of (1c) (or the above-mentioned(1d)), in which cysteines present in the amino acid sequence of theabove-mentioned (1c) (or the above-mentioned (1d)) and the substitutedanother amino acid (preferably, the substituted another amino acid isserine) remain include an amino acid in which cysteines at amino acidnumbers 10, 26, 39, 44, 72, 82, 91, 101, 124, 140, 153, 158, 168, 185,195, 204, 214, 229, 245, 258, 263, 291, 301, 311 and 321 of the aminoacid sequence of the above-mentioned (1c) (or the above-mentioned (1d)),and the substituted another amino acids (preferably, the substitutedanother amino acid is serine) are not changed, and the amino acidsequence parts other than the amino acids have a homology of not lessthan about 85%, preferably not less than about 90%, further preferablynot less than about 95%, most preferably not less than about 98%, withthe amino acid sequence shown in SEQ ID NO: 2 and the like. As usedherein, the “homology” means the same as above.

More preferably, the amino acid sequence substantially the same as theabove-mentioned amino acid sequence of (1c) (or the above-mentioned(1d)), in which cysteines present in the amino acid sequence of theabove-mentioned (1c) (or the above-mentioned (1d)) and the substitutedanother amino acid (preferably, the substituted another amino acid isserine) remain is an amino acid in which cysteines at amino acid numbers10, 26, 39, 44, 72, 82, 91, 101, 124, 140, 153, 158, 168, 185, 195, 204,214, 229, 245, 258, 263, 291, 301, 311 and 321 of the amino acidsequence of the above-mentioned (1c) (or the above-mentioned (1d)), andthe substituted another amino acids (preferably, the substituted anotheramino acid is serine) are not changed, and the amino acid sequence partsother than the amino acids have an identity of not less than about 85%,preferably not less than about 90%, further preferably not less thanabout 95%, most preferably not less than about 98%, with the amino acidsequence shown in SEQ ID NO: 2 and the like.

The position other than cysteines present in the amino acid sequence ofthe above-mentioned (1c) (or the above-mentioned (1d)) and thesubstituted another amino acid (preferably, the substituted anotheramino acid is serine) is the position other than the amino acid numbers10, 26, 39, 44, 72, 82, 91, 101, 124, 140, 153, 158, 168, 185, 195, 204,214, 229, 245, 258, 263, 291, 301, 311 and 321 of the amino acidsequence of the above-mentioned (1c) (or the above-mentioned (1d)), andis not particularly limited as long as it is a position at which amutant mouse AIM containing deletion, addition, insertion orsubstitution, or a combination thereof can maintain or improve theactivities the wild-type mouse AIM has. The activities of wild-typemouse AIM, amino acid used for substitution, number amino acids fordeletion, addition, insertion or substitution or a combination thereof,and the like may be similar to those of the mutant human AIM of thepresent invention.

The mutant human AIM of the present invention and mutant mouse AIM(hereinafter the mutant human AIM of the present invention and mutantmouse AIM are also collectively referred to as the mutant AIM of thepresent invention) may be further added with a signal peptide. Thewild-type human AIM is translated in the cell as an immature wild-typehuman AIM in which a signal peptide shown in SEQ ID NO: 7 is linked tothe N-terminal of the amino acid sequence shown in SEQ ID NO: 1, andconverted to a mature protein by cleavage of the aforementioned signalpeptide when secreted outside the cell. Similarly, wild-type mouse AIMis translated in the cell as an immature wild-type mouse AIM in which asignal peptide shown in SEQ ID NO: 8 is linked to the N-terminal of theamino acid sequence shown in SEQ ID NO: 2, and converted to a matureprotein by cleavage of the aforementioned signal peptide when secretedoutside the cell. Due to the addition of the signal peptide, when mutantAIM is expressed in the cells as a recombinant AIM, the mutant AIM issecreted outside the cell and the recovery is facilitated.

The mutant AIM of the present invention can be produced according to aknown peptide synthesis method.

The peptide synthesis method may be any of, for example, solid phasesynthesis process and solution phase synthesis process. The objectmutant AIM can be produced by condensing a partial peptide or amino acidcapable of constituting the mutant AIM of the present invention and theremaining portion and, when the resultant product has a protectinggroup, removing the protecting group.

Here, the condensation and removal of the protecting group are performedaccording to a method known per se, for example, the methods describedin the following (1) and (2).

(1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York (1966) (2) Schroeder and Luebke, The Peptide,Academic Press, New York (1965)

The thus-obtained mutant AIM of the present invention can be purifiedand isolated by a known purification method. Examples of thepurification method here include solvent extraction, distillation,column chromatography, liquid chromatography, recrystallization, acombination of these and the like.

When the mutant AIM of the present invention obtained by theabove-mentioned method is a free form, the free form can be converted toa suitable salt by a known method or a method analogous thereto.Conversely, when the mutant AIM of the present invention is obtained asa salt, the salt can be converted to a free form or other salt by aknown method or a method analogous thereto.

Furthermore, the mutant AIM of the present invention can also beproduced by culturing a host cell containing an expression vectorcontaining a nucleic acid encoding same, and separating and purifyingthe mutant AIM from the resulting culture. The nucleic acid encoding themutant AIM of the present invention may be DNA or RNA, or may be DNA/RNAchimera. Preferred is DNA. In addition, the nucleic acid may be doublestranded or single stranded. When it is double stranded, it may be adouble stranded DNA, a double stranded RNA or a DNA:RNA hybrid. When itis single stranded, it may be a sense strand (i.e., coding strand), oran antisense strand (i.e., non-coding strand).

As DNA encoding the mutant AIM of the present invention, synthetic DNAand the like can be mentioned. For example, it can be acquired byconverting, according to a method known per se such as ODA-LA PCRmethod, Gapped duplex method, Kunkel method and the like, or a methodanalogous thereto, and by using a known kit, for example, Mutan™-superExpress Km (TAKARA BIO INC.), Mutan™-K (TAKARA BIO INC.) and the like, aprimer for mutation introduction, and a full-length AIM cDNA (e.g., inthe case of human, the base sequence shown in SEQ ID NO: 9 can bementioned, in the case of mouse, the base sequence shown in SEQ ID NO:10 can be mentioned), which was directly amplified by ReverseTranscriptase-PCR (hereinafter abbreviated as “RT-PCR method”) by usingtotal RNA or mRNA fraction prepared from a cell or tissue as a template.Alternatively, it can also be acquired by converting, according to theabove-mentioned method, a cDNA cloned from a cDNA library, prepared byinserting a fragment of the above-mentioned total RNA or mRNA into asuitable vector, by colony or plaque hybridization method or PCR methodand the like. The vector used for the library may be any such asbacteriophage, plasmid, cosmid, phagemid and the like.

The nucleic acid containing a base sequence encoding the mutant AIM ofthe present invention is not limited as long as it is a nucleic acidcontaining a codon corresponding to the amino acid sequence of themutant AIM of the present invention. Examples thereof in the case of amutant human AIM to which a signal peptide is added include a DNAcontaining the same or substantially the same base sequence as the basesequence shown in SEQ ID NO: 11 in which G at base number 572 of thebase sequence shown in SEQ ID NO: 9 is substituted with C, the basesequence shown in SEQ ID NO: 12 in which G at base number 899 of thebase sequence shown in SEQ ID NO: 9 is substituted with C, or the basesequence shown in SEQ ID NO: 13 having both substitutions and the like,in the case of mutant mouse AIM added with signal peptide, it includes aDNA containing a base sequence the same or substantially the same as thebase sequence shown in SEQ ID NO: 14 in which G at base number 581 ofthe base sequence shown in SEQ ID NO: 10 is substituted with C and thelike.

Here, the “substantially the same base sequence” refers to a basesequence having a mutation that does not change the amino acid sequenceencoded by the original base sequence (silent mutation).

An expression vector containing a nucleic acid encoding the mutant AIMof the present invention can be produced, for example, by isolating aDNA fragment encoding the aforementioned mutant AIM, and ligating theDNA fragment at the downstream of a promoter in a suitable expressionvector. For example, an expression vector containing a DNA encoding themutant human AIM of the present invention can be obtained by insertingthe DNA consisting of the base sequence shown in SEQ ID NO: 11 into anexpression vector pCAGGS, and transforming Escherichia coli with theobtained plasmid.

As the expression vector, an animal cell expressing plasmid (e.g.,pCAGGS, pSRα, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo) is used.

The promoter may be any as long as it is an appropriate promoter for thehost used for gene expression.

For example, when the host is an animal cell, β-actin promoter, SRαpromoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter,RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemiavirus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, trcpromoter, trc modified promoter and the like are used.

As the expression vector, one containing, when desired, enhancer,splicing signal, poly A addition signal, selection marker, SV40replication origin (hereinafter sometimes to be abbreviated as SV40 ori)and the like can be used besides those mentioned above. Examples of theselection marker include dihydrofolate reductase gene (hereinaftersometimes to be abbreviated as dhfr, methotrexate (MTX) resistance),neomycin resistance gene (hereinafter sometimes to be abbreviated asneo^(r), G418 resistance) and the like. Particularly, when dhfr genedeficient Chinese hamster cell is used, and dhfr gene is used as aselection marker, the object gene can also be selected in a medium freeof thymidine.

The mutant AIM can be produced by introducing an expression vectorcontaining the above-mentioned nucleic acid encoding the mutant AIM intoa host cell, and culturing the obtained host cell.

As the host cell, an animal cell is preferable for the mutant AIM of thepresent invention.

As the animal cell, for example, cells such as COS-7, Vero, CHO, CHO(dhfr⁻), CHO-K1, CHO-S, L, AtT-20, GH3, FL, HEK293, NIH3T3, Balb3T3,FM3A, L929, SP2/0, P3U1, B16, P388 and the like are used.

Gene transfer can be performed according to a known method.

Gene can be introduced into the animal cell according to the methoddescribed in, for example, Cell Engineering, extra issue 8, New CellEngineering Experimental Protocol, 263-267 (1995) (published byShujunsha), Virology, vol. 52, 456 (1973).

A transfected host cell can be cultured according to a known method.

When the host cell is an animal cell, the medium used for culture ispreferably minimum essential medium (MEM), Dulbecco's modified Eaglemedium (DMEM), RPMI 1640 medium, 199 medium or the like, each containingabout 5-about 20% of fetal bovine serum. The pH of the medium ispreferably about 6-about 8. The transformant is cultured generally atabout 30-about 40° C. for about 15-about 60 hr. Where necessary,aeration and stirring may be performed.

As mentioned above, the mutant AIM of the present invention can beproduced intracellularly or extracellularly using the host cell.

The mutant AIM of the present invention can be separated and purified bya method known per se from the culture obtained by culturing theaforementioned transfected host cell.

For example, when the mutant AIM of the present invention is extractedfrom cytoplasm of the host cell, a method including suspending the hostcells collected from a culture by a known method in a suitable buffer,rupturing the host cells by ultrasonication, lysozyme and/orfreeze-thawing and the like, and obtaining a crude extract of a solubleprotein by centrifugation, filtration and the like as appropriate. Thebuffer may contain protein denaturant such as urea, hydrochloric acidguanidine and the like, and surfactant such as TritonX-100™ and thelike. When the mutant AIM of the present invention is secreted out fromthe cell, a method for separating the culture supernatant from theculture by centrifugation, filtration and the like, and the like isused.

The mutant AIM of the present invention contained in the thus-obtainedsoluble fraction and culture supernatant can be isolated and purified bya method known per se. As such method, a method utilizing the solubilitysuch as salting out, solvent precipitation and the like; a method mainlyutilizing difference in the molecular weight such as dialysis,ultrafiltration, gel filtration method, and SDS-polyacrylamide gelelectrophoresis and the like; a method utilizing difference in theelectric charge such as ion exchange chromatography and the like; amethod utilizing specific affinity such as affinity chromatography andthe like; a method utilizing difference in the hydrophobicity such asreversed-phase high performance liquid chromatography and the like; amethod utilizing difference in isoelectric point such as isoelectricfocusing and the like; a method using an antibody, and the like areused. These methods can also be combined as appropriate.

The presence of the thus-obtained mutant AIM of the present inventioncan be confirmed by enzyme immunoassay, Western blotting and the like,using an antibody to the mutant AIM.

The present invention also provides a pharmaceutical compositioncontaining the mutant AIM of the present invention (hereinafter to bealso referred to as the pharmaceutical composition of the presentinvention). The inventions heretofore reported that AIM can be used forthe prophylaxis or treatment of obesity, hepatic diseases, and renaldiseases (WO 2010/140531, WO 2013/162021, WO 2015/119253). The mutantAIM of the present invention also maintains or improves the activitiessimilar to those of wild-type AIM, it can also be used for theprophylaxis or treatment of obesity, hepatic diseases, and renaldiseases.

Examples of the subject of administration of the pharmaceuticalcomposition of the present invention include humans and otherwarm-blooded animals (e.g., mouse, rat, rabbit, sheep, swine, bovine,cat, dog, monkey, avian and the like).

The hepatic diseases to be the application target of the pharmaceuticalcomposition of the present invention include, for example, fatty liver,non-alcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. Therenal diseases to be the application target of the pharmaceuticalcomposition of the present invention include, for example, acute renalfailure, chronic nephritis, chronic renal failure, nephrotic syndrome,diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensivenephropathy, and nephropathy associated with collagen disease or IgMnephropathy, and acute renal failure or chronic renal failure ispreferred. The nephropathy associated with collagen disease isrepresented by, for example, lupus nephritis.

The pharmaceutical composition of the present invention is of lowtoxicity, and can be administered as a liquid as it is, or as anappropriate dosage form of pharmaceutical composition, to humans orother warm-blooded mammals (e.g., mouse, rat, rabbit, sheep, swine,bovine, cat, dog, monkey, avian and the like) orally or parenterally(e.g., intravascular administration, subcutaneous administration and thelike).

As examples of the pharmaceutical composition for parenteraladministration, injections, suppositories and the like are used; theinjections may include dosage forms such as intravenous injections,subcutaneous injections, intracutaneous injections, intramuscularinjections and drip infusion injections. Such an injection can beprepared according to a publicly known method. An injection can beprepared by, for example, dissolving, suspending or emulsifying theabove-mentioned mutant human AIM of the present invention in a sterileaqueous or oily solution in common use for injections. As examples ofaqueous solutions for injection, physiological saline, an isotonicsolution comprising glucose or another auxiliary drug, and the like canbe used, which may be used in combination with an appropriatesolubilizer, for example, alcohol (e.g., ethanol), polyalcohol (e.g.,propylene glycol, polyethylene glycol), non-ionic surfactant [e.g.,polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenatedcastor oil)] and the like. As examples of oily solutions, sesame oil,soybean oil and the like can be used, which may be used in combinationwith benzyl benzoate, benzyl alcohol and the like as solubilizers. Theprepared injection solution is preferably filled in an appropriateampoule. Suppositories used for rectal administration may be prepared bymixing the above-mentioned mutant AIM with an ordinary suppository base.

As the pharmaceutical composition for oral administration, solid orliquid dosage forms, specifically tablets (including sugar-coated tablesand film-coated tablets), pills, granules, powders, capsules (includingsoft capsules), syrups, emulsions, suspensions and the like can bementioned. Such a pharmaceutical composition is produced by a publiclyknown method, and may comprise a carrier, diluent or excipient in commonuse in the field of pharmaceutical making. As examples of the carrier orexcipient for tablets, lactose, starch, sucrose, magnesium stearate andthe like can be used.

While the dose of the pharmaceutical composition of the presentinvention varies depending on the subject of administration, targetdisease, symptoms, administration route and the like, for example, whenused for an adult, the mutant AIM of the present invention isconveniently administered by intravenous injection at typically about0.01-20 mg/kg body weight, preferably about 0.1-10 mg/kg body weight,further preferably about 0.1-5 mg/kg body weight, as one dose, about 1-5times/day, preferably about 1-3 times/day, for about 1-21 days,preferably about 1-14 days. In the case of other parenteraladministrations and oral administrations, doses similar thereto can beadministered. When the symptoms are particularly severe, the dose may beincreased according to the symptoms.

The pharmaceutical composition of the present invention may contain anyother active ingredients that do not produce an unwanted interactionwhen formulated with the mutant AIM of the present invention.

EXAMPLE

The present invention is explained more specifically in the following byreferring to Examples and Reference Examples, which are not to beconstrued as limitative.

Example 1: Formation of Wild-Type Human Recombinant AIM (rAIM) Multimer

The wild-type human rAIM was prepared as follows. That is, human AIMstable expression strain obtained by introducing pCAGGS-human AIMexpression vector into HEK293 was cultured in Dulbecco's Modified EagleMedium (DMEM, Invitrogen) containing 5% FBS, Glutamax, gentamicin for 3days, and the culture supernatant was recovered. The recovered culturesupernatant was applied to an antibody column obtained by immobilizingmouse anti-human AIM monoclonal antibody (clone 7, autologouslymanufactured) on HiTrap NHS-activated HP column (GE Healthcare LifeSciences) to purify human rAIM. The human rAIM bound to the antibodycolumn was eluted with 0.1 M glycine-HCl (pH2.5), and immediatelyneutralized with 1 M Tris-HCl (pH 8.5) to obtain human rAIM eluate. Thebuffer in the eluate was substituted with Dulbecco's Phosphate-BufferedSaline (DPBS) by using Amicon Ultra filter concentrators (Millipore) toperform concentration. The protein level of the human rAIM concentratewas quantified using Bicinchoninic acid (BCA) assay (Pierce), and thefinal concentration was adjusted to 2.0 mg/mL with DPBS. The wild-typehuman rAIM protein (1 μg) purified and concentrated as mentioned abovewas separated under non-reducing conditions by polyacrylamide gelelectrophoresis (SDS-PAGE) based on the difference in the molecularweights, Coomassie Brilliant Blue (CBB) staining was performed, andwild-type human rAIM was detected.

As a result, when human rAIM was purified from the human AIM stableexpression strain and prepared as a high concentration solution, humanrAIM formed a multimer (FIG. 1 ). Particularly, precipitation of some ofthe multimers having a large molecular weight (complex in which a largeamount of rAIM is multimerized: multimeric rAIM corresponding to thehigh molecular weight band in lane A of FIG. 1 and a multimeric rAIMremaining in the slot) was feared. This suggests a risk that, when humanrAIM is purified and administered to a living body as a therapeuticdrug, the precipitated multimers may block fine blood vessels and leadto a serious accident. In view of the possible clinical application ofhuman rAIM in the future, creation of a mutant human AIM that does notform at least a multimer having a large molecular weight is desired.

Example 2: Formation of Wild-Type Mouse Recombinant AIM (rAIM) Multimer

The wild-type mouse rAIM was prepared as follows. That is, mouse AIMstable expression strain obtained by introducing pCAGGS-mouse AIMexpression vector into HEK293 was cultured in DMEM containing 5% FBS,Glutamax, gentamicin for 3 days, and the culture supernatant wasrecovered. The recovered culture supernatant was applied to an antibodycolumn obtained by immobilizing rat anti-mouse AIM monoclonal antibody(clone 36, autologously manufactured) on HiTrap NHS-activated HP columnto purify mouse rAIM. The mouse rAIM bound to the antibody column waseluted with 0.1 M glycine-HCl (pH2.3), and immediately neutralized with1 M Tris-HCl (pH 8.5) to obtain mouse rAIM eluate. The buffer in theeluate was substituted with DPBS by using Amicon Ultra filterconcentrators to perform concentration. The protein level of the mouserAIM concentrate was quantified using BCA assay, and the finalconcentration was adjusted to 2.0 mg/mL with DPBS. The wild-type mouserAIM protein (1 μg) purified and concentrated as mentioned above wasseparated under non-reducing conditions by SDS-PAGE based on thedifference in the molecular weights, CBB staining was performed, andwild-type mouse rAIM was detected.

As a result, mouse rAIM did not form a multimer like human rAIM, andmonomers and dimers alone were present (FIG. 2 ).

Example 3: Prediction of Three-Dimensional Structure of Wild-Type Humanand Mouse AIMs

The wild-type human AIM has 8, 9, 9 cysteines in three SRCR domains(SRCR1, SRCR2, SRCR3), respectively (FIG. 3 ). A signal peptide islinked to the N-terminal of immature AIM and the immature AIM isconverted to a mature protein by cleavage of the signal peptide whensecreted outside the cell. To verify the possibility of theabove-mentioned disulfide bond due to cysteine present in human AIMbeing involved in multimerization, the present inventors predicted thethree-dimensional structure of human AIM. The three-dimensionalstructure of human AIM was predicted by two-step modeling of the partialstructure shown by three SRCR domains and the entire structure includingthe three SRCR domains and the hinge (underlined in FIG. 3 ). ASwiss-Model server (http://swissmodel.expasy.org/SWISS-MODEL) was usedfor homology modeling for each SRCR domain. As a result of individualsequence homology search by Blast and HHBlits on the three SRCR domainsof human AIM, human CD6 (5a2e.1.A; PDB ID: 5A2E) which belongs to SRCRsuperfamily Group B to which human AIM belongs similarly showed goodsequence homology of not less than 30% to all SRCR domains. Therefore,using human CD6 as a template, three-dimensional structures of threeSRCR domains were constructed by Promod-II6. Next, using Prime version4.2 (Schrodinger, LLC, New York, N.Y., 2015), the entire structure ofhuman AIM was constructed by introducing a sequence (hinge) other thanthe SRCR domain. The three-dimensional structure of the entire human AIMwas obtained using the molecular dynamics calculation (310 K, 20 ns) byDesmond with OPLS_2005 as a force field, and equilibrating the entirestructure of the human AIM in a periodic box model filled with watermolecules (SPC). The four disulfide bonds in each SRCR domain of humanAIM were reproduced using the SRCR domain of human CD6 as a template.The results of prediction of the three-dimensional structure ofwild-type human AIM are shown in FIG. 4 . It was found that 8 cysteinesof the 8, 9, 9 cysteines respectively present in the three SRCR domainsof the wild-type human AIM are used for the disulfide bonds inrespective SRCR domains. However, the inventors have found thatwild-type human AIM contains one isolated cysteine (Solitary Cys) ineach of SRCR2 domain and SRCR3 domain, namely, the cysteine at aminoacid number 168 and the cysteine at amino acid number 277 of wild-typehuman AIM shown in SEQ ID NO: 1.

In addition, the inventors predicted the three-dimensional structure ofmouse AIM by a similar method. The results of prediction of thethree-dimensional structure of wild-type mouse AIM are shown in FIG. 5 .The wild-type mouse AIM has 8, 9, 8 cysteines in respective SRCR domains(FIG. 6 ). It was found that 8 cysteines of those cysteines are used forthe disulfide bonds in respective SRCR domains. However, the inventorshave found that wild-type mouse AIM contains an isolated cysteine(Solitary Cys) in SRCR2 domain, namely, the cysteine at amino acidnumber 168 of wild-type mouse AIM shown in SEQ ID NO: 2.

Example 4: Formation of Mutant Human rAIM Multimer

To verify the possibility of the cysteine at amino acid number 168 andthe cysteine at amino acid number 277 of wild-type human AIM shown inSEQ ID NO: 1 being involved in the multimerization of wild-type humanrAIM, the inventors prepared mutant human rAIM in which theabove-mentioned cysteines are substituted with different amino acids.First, to maintain the function of wild-type human rAIM, the amino acidto be used for substitution was studied. Since cysteine is hydrophilic(hydrogen bond can be formed), electric charge 0, and non-aromatic,serine, threonine, asparagine, glutamine were selected as 4 similaramino acids. By comparison of molecular shape and size, since serine hada structure and properties closest to those of cysteine from among theabove-mentioned 4 amino acids, serine was selected as amino acid to beused for substitution. The mutant human rAIM was prepared as follows.That is, pCAGGS-human AIM-2CS, pCAGGS-human AIM-3CS, and pCAGGS-humanAIM-2/3CS expression vectors that respectively express mutant human AIM(hereinafter to be also referred to as “2CS” in Examples 4, 6, 7) (SEQID NO: 3) obtained by substituting codon TGC encoding the cysteine atamino acid number 168 of wild-type human AIM shown in SEQ ID NO: 1 withTCC, mutant human AIM (hereinafter to be also referred to as “3CS”) (SEQID NO: 4) obtained by substituting codon TGC encoding the cysteine atamino acid number 277 of wild-type human AIM shown in SEQ ID NO: 1 withTCC, and mutant human AIM having both of the aforementionedsubstitutions (hereinafter to be also referred to as “2/3CS”) (SEQ IDNO: 5) were respectively produced. Then, the expression vector wasintroduced into HEK293T cells by an electroporation method, and eachmutant human rAIM expressed transiently by culturing for 3 days wasrecovered from the culture supernatant. Thereafter, each mutant humanrAIM was purified and concentrated using an antibody column by a methodsimilar to that in Example 1. The purified and concentrated wild-type,2CS, 3CS, 2/3CS human rAIM proteins, each 100 ng, were separated bySDS-PAGE under non-reducing conditions based on the difference in themolecular weights, Oriole staining was performed, and each human rAIMprotein was detected.

As a result, 2CS and 3CS obtained by substituting one isolated cysteinepresent in each of the SRCR2 domain and SRCR3 domain with serine did notform a multimer but formed monomers and dimers alone, like wild-typemouse AIM (FIG. 7 ). In addition, 2/3CS obtained by co-substituting theabove-mentioned isolated cysteine with serine formed monomer alone (FIG.7 ). Therefore, it was found that the multimerization of wild-type humanrAIM was caused by the cysteine at amino acid number 168 and thecysteine at amino acid number 277 of wild-type human AIM shown in SEQ IDNO: 1. The recovery rate of the final purified rAIM (excludingprecipitates from wild-type human rAIM) from the culture supernatant was32, 52, 53, 61% (average of the recovery rate after 3 times of transientexpression and purification) in the order of wild-type, 2CS, 3CS,2/3CS), and the recovery rate increased in the mutant type.

Example 5: Formation of Mutant Mouse rAIM Dimer

To verify the possibility of the cysteine at amino acid number 168 ofwild-type mouse AIM shown in SEQ ID NO: 2 being involved in dimerizationof wild-type mouse rAIM, the inventors prepared mutant mouse rAIM inwhich the above-mentioned cysteine is substituted with serine, as inExample 4. The mutant mouse rAIM was prepared as follows. That is,pCAGGS-mouse AIM-2CS expression vector that expresses mutant mouse AIM(hereinafter to be also referred to as “2CS” in Examples 5, 7) (SEQ IDNO: 6) obtained by substituting codon TGT encoding the cysteine at aminoacid number 168 of wild-type mouse AIM shown in SEQ ID NO: 2 with TCTwas produced. Then, the expression vector was introduced into HEK293Tcells by an electroporation method, and mutant mouse rAIM expressedtransiently by culturing for 3 days was recovered from the culturesupernatant. Thereafter, mutant mouse rAIM was purified and concentratedusing an antibody column by a method similar to that in Example 2. Thepurified and concentrated wild-type, 2CS mouse rAIM proteins, each 100ng, were separated by SDS-PAGE under non-reducing conditions based onthe difference in the molecular weights, Oriole staining was performed,and each mouse rAIM protein was detected.

As a result, 2CS obtained by substituting one isolated cysteine presentin the SRCR2 domain with serine formed only a monomer and did not form adimer (FIG. 8 ). Therefore, it was found that dimerization of wild-typemouse rAIM is caused by the cysteine at amino acid number 168 ofwild-type mouse AIM shown in SEQ ID NO: 2.

Example 6: Function of Mutant Human rAIM

AIM is taken up by various cells such as macrophages, hepatocytes andadipocytes, and exerts various actions on respective cells. Theinventors verified using macrophage cells whether each mutant human rAIM(2CS, 3CS, and 2/3C) lost the function taken up by the above-mentionedcells by mutation. FITC labeling was performed on wild-type and eachmutant (2CS, 3CS, and 2/3C) human rAIM usingfluorescein-4-isothiocyanate (Dojindo Laboratories). The labeling rateof each human rAIM was similar. Thereafter, each human rAIM wasco-cultured for 30 min with F4/80-positive macrophages isolated from theabdominal cavity of AIM-deficient mice in DMEM containing 5% FBS at aconcentration of 20 μg/mL, and the human rAIM uptake reaction wasperformed. The cells were collected and washed, the intracellular uptakeof each rAIM was analyzed by measuring the FITC mean fluorescenceintensity in F4/80-positive macrophages by a flow cytometer (BDFACSCelesta).

As a result, the uptake of each mutant human rAIM into macrophages wasnot attenuated as compared to wild-type human rAIM. Therefore, it isconsidered that each mutant human rAIM was not functionally attenuated.On the contrary, interestingly, 2CS was uptaken by macrophages more thanwild-type AIM (FIG. 9 ).

Example 7: Formation of Complex of Wild-Type or Mutant rAIM and IgMPentamer

HEK293T cells forcibly expressing IgM and J chain, and HEK293T cellsforcibly expressing wild type or mutant (2CS) rAIM were co-cultured for24 hr, and the proteins contained in the culture supernatant wereseparated by SDS-PAGE under non-reducing conditions. Then, the proteinseparated on the membrane filter was transcribed, and whether rAIM andIgM pentamer formed a complex was verified using anti-AIM (a-AIM)antibody or anti-IgM (a-IgM) antibody (mouse (FIG. 10A), human (FIG.10B)). As a result, in both mouse and human, wild-type rAIM bound toIgM, but 2CS did not bind to IgM.

INDUSTRIAL APPLICABILITY

The mutant AIM of the present invention has the function equivalent tothat of the wild-type AIM, or has an improved function, and ischaracterized in that it does not multimerize when expressed as arecombinant AIM. By the absence of multimerization, recombinant AIM doesnot precipitate by insolubilization and, as a result, the recovery rateof recombinant AIM is improved and the risk associated with in vivoadministration is avoided. Furthermore, when administered to a livingbody, the mutant AIM of the present invention does not form a complexwith an IgM pentamer and thus is not inactivated, and can prevent adecrease in the titer. This application is based on a patent applicationNo. 2017-220733 filed in Japan (filing date: Nov. 16, 2017), thecontents of which are incorporated in full herein.

1. A mutant human AIM comprising an amino acid sequence of any one ofthe following (1) to (5) and having an activity of wild-type human AIM(1) an amino acid sequence wherein cysteine at amino acid number 168 ofthe amino acid sequence shown in SEQ ID NO: 1 is substituted withanother amino acid, (2) an amino acid sequence wherein cysteine at aminoacid number 277 of the amino acid sequence shown in SEQ ID NO: 1 issubstituted with another amino acid, (3) an amino acid sequence whereincysteine at amino acid number 168 of the amino acid sequence shown inSEQ ID NO: 1 is substituted with another amino acid, and an amino acidsequence wherein cysteine at amino acid number 277 of the amino acidsequence shown in SEQ ID NO: 1 is substituted with another amino acid,(4) an amino acid sequence substantially the same as the amino acidsequence of any one of (1) to (3), and cysteines present in the aminoacid sequence of any one of (1) to (3) and the substituted another aminoacid remain, (5) an amino acid sequence further comprising deletion,addition, insertion or substitution of one to several amino acids or acombination thereof at a position other than cysteines present in theamino acid sequence of any one of (1) to (3) and the substituted anotheramino acid.
 2. The mutant human AIM according to claim 1, wherein saidanother amino acid is an amino acid selected from the group consistingof asparagine, glutamine, serine, and threonine.
 3. The mutant human AIMaccording to claim 1, wherein said another amino acid is serine.
 4. Amutant mouse AIM comprising an amino acid sequence of any one of thefollowing (1) to (3) and having an activity of wild-type mouse AIM (1)an amino acid sequence wherein cysteine at amino acid number 168 of theamino acid sequence shown in SEQ ID NO: 2 is substituted with anotheramino acid, (2) an amino acid sequence substantially the same as theamino acid sequence of (1), and cysteines present in the amino acidsequence of (1) and the substituted another amino acid remain, (3) anamino acid sequence further comprising deletion, addition, insertion orsubstitution of one to several amino acids or a combination thereof at aposition other than cysteines present in the amino acid sequence of (1)and the substituted another amino acid.
 5. The mutant mouse AIMaccording to claim 4, wherein said another amino acid is an amino acidselected from the group consisting of asparagine, glutamine, serine, andthreonine.
 6. The mutant mouse AIM according to claim 4, wherein saidanother amino acid is serine.
 7. A nucleic acid comprising the basesequence of the following (1) or (2) (1) a base sequence encoding themutant human AIM according to any one of claims 1 to 3, (2) a basesequence encoding the mutant mouse AIM according to any one of claims 4to
 6. 8. An expression vector comprising the nucleic acid according toclaim
 7. 9. A host cell comprising the expression vector according toclaim
 8. 10. A method for producing a mutant human AIM or a mutant mouseAIM, comprising culturing the host cell according to claim
 9. 11. Apharmaceutical composition comprising a mutant AIM of the following (1)or (2) (1) the mutant human AIM according to any one of claims 1 to 3,(2) the mutant mouse AIM according to any one of claims 4 to 6.